Implementation of a Novel Method for Processing Proteins from Acetic Acid Bacteria via Liquid Chromatography Coupled with Tandem Mass Spectrometry.

Acetic acid bacteria (AAB) and other members of the complex microbiotas, whose activity is essential for vinegar production, display biodiversity and richness that is difficult to study in depth due to their highly selective culture conditions. In recent years, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has emerged as a powerful tool for rapidly identifying thousands of proteins present in microbial communities, offering broader precision and coverage. In this work, a novel method based on LC-MS/MS was established and developed from previous studies. This methodology was tested in three studies, enabling the characterization of three submerged acetification profiles using innovative raw materials (synthetic alcohol medium, fine wine, and craft beer) while working in a semicontinuous mode. The biodiversity of existing microorganisms was clarified, and both the predominant taxa (Komagataeibacter, Acetobacter, Gluconacetobacter, and Gluconobacter) and others never detected in these media (Asaia and Bombella, among others) were identified. The key functions and adaptive metabolic strategies were determined using comparative studies, mainly those related to cellular material biosynthesis, energy-associated pathways, and cellular detoxification processes. This study provides the groundwork for a highly reliable and reproducible method for the characterization of microbial profiles in the vinegar industry.

Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis.

Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.

Effect of pH on the denitrification proteome of the soil bacterium Paracoccus denitrificans PD1222.

Denitrification is a respiratory process by which nitrate is reduced to dinitrogen. Incomplete denitrification results in the emission of the greenhouse gas nitrous oxide and this is potentiated in acidic soils, which display reduced denitrification rates and high N2O/N2 ratios compared to alkaline soils. In this work, impact of pH on the proteome of the soil denitrifying bacterium Paracoccus denitrificans PD1222 was analysed with nitrate as sole energy and nitrogen source under anaerobic conditions at pH ranging from 6.5 to 7.5. Quantitative proteomic analysis revealed that the highest difference in protein representation was observed when the proteome at pH 6.5 was compared to the reference proteome at pH 7.2. However, this difference in the extracellular pH was not enough to produce modification of intracellular pH, which was maintained at 6.5 ± 0.1. The biosynthetic pathways of several cofactors relevant for denitrification and nitrogen assimilation like cobalamin, riboflavin, molybdopterin and nicotinamide were negatively affected at pH 6.5. In addition, peptide representation of reductases involved in nitrate assimilation and denitrification were reduced at pH 6.5. Data highlight the strong negative impact of pH on NosZ synthesis and intracellular copper content, thus impairing active NosZ assembly and, in turn, leading to elevated nitrous oxide emissions.

Meta-omic evaluation of bacterial microbial community structure and activity for the environmental assessment of soils: overcoming protein extraction pitfalls.

Microorganisms play unique, essential and integral roles in the biosphere. This work aims to assess the utility of soil's metaomics for environmental diagnosis. Doñana National Park (DNP) was selected as a natural lab since it contains a strictly protected core that is surrounded by numerous threats of pollution. Culture-independent high-throughput molecular tools were used to evaluate the alterations of the global structure and metabolic activities of the microbiome. 16S rRNA sequencing shows lower bacterial abundance and diversity in areas historically exposed to contamination that surround DNP. For metaproteomics, an innovative post-alkaline protein extraction protocol was developed. After NaOH treatment, successive washing with Tris-HCl buffer supplemented with glycerol was essential to eliminate interferences. Starting from soils with different physicochemical characteristics, the method renders proteins with a remarkable resolution on SDS-PAGE gels. The proteins extracted were analysed by using an in-house database constructed from the rRNA data. LC-MS/MS analysis identified 2182 non-redundant proteins with 135 showing significant differences in relative abundance in the soils around DNP. Relevant global biological processes were altered in response to the environmental changes, such as protective and antioxidant mechanisms, translation, folding and homeostasis of proteins, membrane transport and aerobic respiratory metabolism.

Constructing a de novo transcriptome and a reference proteome for the bivalve Scrobicularia plana: Comparative analysis of different assembly strategies and proteomic analysis.

Scrobicularia plana is a coastal and estuarine bivalve widely used in ecotoxicological studies. However, the underlying molecular mechanisms for S. plana pollutant responses are hardly known due to the lack of molecular databases. Thus, in this study we present a holistic approach to assess a robust reference transcriptome and proteome of this clam. A mixture of control and metal-exposed individuals was used for mRNA isolation. Four sets of high quality filtered preprocessed reads were generated (two quality scores and two sequenced lengths) and assembled with Mira, Ray and Trinity algorithms. The sixty-four generated assemblies were refined, filtered and evaluated for their proteomic quality. Eight assemblies presented top Detonate scores but one was selected due to its compactness and biological representation, which was generated: (i) from the highest quality dataset (Q20L100), (ii) using Trinity algorithm with all k-mers (AtKa), (iii) removing redundancy by CD-HIT (RR80), and (iv) filtering out poor contigs (F), that was subsequently named Q20L100AtKaRR80F. S. plana proteomic analysis revealed 10,017 peptide groups that corresponded to 2066 proteins with a wide coverage of molecular functions and biological processes, confirming the strength of the database generated.

Effects of Erythrodiol on the Antioxidant Response and Proteome of HepG2 Cells.

Erythrodiol (EO) is a pentacyclic triterpenic alcohol found in olive tree leaves and olive oil, and it has important effects on the health properties and quality of olive oil. In this study, we characterized the cytotoxic effects of EO on human hepatocarcinoma (HepG2) cells by studying changes in cell viability, reactive oxygen species (ROS) production, antioxidant defense systems, and the proteome. The results reveal that EO markedly decreased HepG2 cell viability without changing ROS levels. The concentrations of glutathione and NADPH were significantly reduced, with selective changes in the activity of several antioxidant enzymes: glutathione peroxidase, glutathione reductase, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. Proteomic data reveal that EO led to the complete elimination or decreased abundance of 41 and 3 proteins, respectively, and the abundance of 29 proteins increased. The results of functional enrichment analysis show that important metabolic processes and the nuclear transport of mature mRNA were impaired, whereas AMP biosynthesis and cell cycle G2/M phase transition were induced. The transcription factors and miRNAs involved in this response were also identified. These potent antiproliferative effects make EO a good candidate for the further analysis of its hepatic antitumor effects in in vivo studies.

Development of New Antiproliferative Compound against Human Tumor Cells from the Marine Microalgae Nannochloropsis gaditana by Applied Proteomics.

Proteomics is a crucial tool for unravelling the molecular dynamics of essential biological processes, becoming a pivotal technique for basic and applied research. Diverse bioinformatic tools are required to manage and explore the huge amount of information obtained from a single proteomics experiment. Thus, functional annotation and protein-protein interactions are evaluated in depth leading to the biological conclusions that best fit the proteomic response in the system under study. To gain insight into potential applications of the identified proteins, a novel approach named "Applied Proteomics" has been developed by comparing the obtained protein information with the existing patents database. The development of massive sequencing technology and mass spectrometry (MS/MS) improvements has allowed the application of proteomics nonmodel microorganisms, which have been deeply described as a novel source of metabolites. Between them, Nannochloropsis gaditana has been pointed out as an alternative source of biomolecules. Recently, our research group has reported the first complete proteome analysis of this microalga, which was analysed using the applied proteomics concept with the identification of 488 proteins with potential industrial applications. To validate our approach, we selected the UCA01 protein from the prohibitin family. The recombinant version of this protein showed antiproliferative activity against two tumor cell lines, Caco2 (colon adenocarcinoma) and HepG-2 (hepatocellular carcinoma), proving that proteome data have been transformed into relevant biotechnological information. From Nannochloropsis gaditana has been developed a new tool against cancer-the protein named UCA01. This protein has selective effects inhibiting the growth of tumor cells, but does not show any effect on control cells. This approach describes the first practical approach to transform proteome information in a potential industrial application, named "applied proteomics". It is based on a novel bioalgorithm, which is able to identify proteins with potential industrial applications. From hundreds of proteins described in the proteome of N. gaditana, the bioalgorithm identified over 400 proteins with potential uses; one of them was selected as UCA01, "in vitro" and its potential was demonstrated against cancer. This approach has great potential, but the applications are potentially numerous and undefined.

Optimizing Shotgun Proteomics Analysis for a Confident Protein Identification and Quantitation in Orphan Plant Species: The Case of Holm Oak (Quercus ilex).

The proteomics of orphan, unsequenced, and recalcitrant organisms is highly challenging. This is the case of the typical Mediterranean forest tree Holm oak (Quercus ilex). Proteomics has moved on quite fast from the classical 2DE-MS to shotgun or gel-free/label-free approaches, with the latter possessing a series of advantages over the gel-based ones. Before translating proteomics data into biological knowledge, a few questions as to the analysis technique itself have to be answered including its confidence in protein identification and quantification. It is important to clearly differentiate a hit from an ortholog and gene product identification, with the difference depending on the database and the confidence parameters (score, number of peptides, and coverage). With respect to quantification and for comparative purposes it is important to make sure that we are within the linear dynamic range. For that, a calibration curve based on mass spectrometry analysis of a serial dilution of the extracts should be performed. Thus, just by validating our data with the aim of improving the quality of the analysis enables us to give a correct interpretation of our results. We show a method that aims to improve the confidence in protein identification and quantification in the orphan species Q. ilex using a shotgun proteomics approach.

Quantitative Analysis of Target Peptides Related to Resistance Against Ascochyta Blight (Peyronellaea pinodes) in Pea.

Peyronellaea pinodes causes Ascochyta blight, one of the major diseases in pea worldwide. Cultivated pea plants have a low resistance to this disease. Although quantitative trait loci (QTLs) involved in the resistance to Ascochyta blight have been identified, the specific genes associated with these QTLs remain unknown, which makes marker-assisted selection difficult. Complex traits alter proteins and their abundance. Quantitative estimation of proteins in pea might therefore be useful in selecting potential markers for breeding. In this work, we developed a strategy using a combination of shotgun proteomics (viz., high performance liquid chromatography-mass spectrometry data-dependent acquisition) and data-independent acquisition (DIA) analysis, to identify putative protein markers associated with resistance to Ascochyta blight and explored its use for breeding selection. For this purpose, an initial list of target peptides based on proteins closely related to resistance to P. pinodes was compiled by using two genotypes with contrasting responses to the disease. Then, targeted data analysis (viz., shotgun proteomics-DIA) was used for constitutive quantification of the target peptides in a representative number of the recombinant inbred line population segregated for resistance as derived from a cross between the two genotypes. Finally, a peptide panel of potential markers for resistance to P. pinodes was built. The results thus obtained are discussed and compared with those of previous gene expression studies using the same parental pea genotypes responding to the pathogen. Also, a molecular defense mechanism against Ascochyta blight in pea is proposed. To the authors' knowledge, this is the first time a targeted proteomics approach based on data analysis has been used to identify peptides associated with resistance to this disease.

An "omic" approach to Pyrocystis lunula: New insights related with this bioluminescent dinoflagellate.

Pyrocystis lunula (Schutt) is a photoautotrophic dinoflagellate without armored form, frequently found in marine environments. Today, there are several biotechnological applications derived from the bioluminescent system of this species. From a post-genomic perspective, in order to have a starting point for studying the proteome of P. lunula, an "omics" approach (transcriptomics-proteomics) was assessed using fresh microalgae samples. A total of 80,874,825 raw reads were generated (11,292,087,505 bp; 55.82% GC) by mRNA sequencing. Very high-quality sequences were assembled into 414,295 contigs (219,203,407 bp; 55.38% GC) using Trinity software, generating a comprehensive reference transcriptome for this species. Then, a P. lunula proteome was inferred and further employed for its analysis on this species. A total of 17,461 peptides were identified, yielding 3182 protein identification hits, including 175 novel proteins. The identified proteins were further categorized according to functional description and gene ontology classification. SIGNIFICANCE: The major contribution of the present work is making available a reference transcriptome and proteome of P. lunula, that is now accessible for the research community, and a functional description of the 3182 proteins inferred from the transcriptome, including 175 novel proteins, which have already been deposited in the ProteomeXchange and NCBI SRA databases, respectively. In addition to this, a series of important factors related to the bioluminescent system and the regulation of gene expression, were identified and described.

Redox and global interconnected proteome changes in mice exposed to complex environmental hazards surrounding Doñana National Park.

Natural environments are receiving an increasing number of contaminants. Therefore, the evaluation and identification of early responses to pollution in these complex habitats is an urgent and challenging task. Doñana National Park (DNP, SW Spain) has been widely used as a model area for environmental studies because, despite its strictly protected core, it is surrounded by numerous threat sources from agricultural, mining and industrial activities. Since many pollutants often induce oxidative stress, redox proteomics was used to detect redox-based variations within the proteome of Mus spretus mice captured in DNP and the surrounding areas. Functional analysis showed that most differentially oxidized proteins are involved in the maintenance of homeostasis, by eliciting mechanisms to respond to toxic substances and oxidative stress, such as antioxidant and biotransformation processes, immune and inflammatory responses, and blood coagulation. Furthermore, changes in the overall protein abundance were also analysed by label-free quantitative proteomics. The upregulation of phase I and II biotransformation enzymes in mice from Lucio del Palacio may be an alert for organic pollution in the area located at the heart of DNP. Metabolic processes involved in protein turnover (proteolysis, amino acid catabolism, new protein biosynthesis and folding) were activated in response to oxidative damage to these biomolecules. Consequently, aerobic respiratory metabolism increased to address the greater ATP demands. Alterations of cholesterol metabolism that could cause hepatic steatosis were also detected. The proteomic detection of globally altered metabolic and physiological processes offers a complete view of the main biological changes caused by environmental pollution in complex habitats.

Proteomic analysis of goat milk kefir: Profiling the fermentation-time dependent protein digestion and identification of potential peptides with biological activity.

Kefir is a fermented dairy product, associated to health benefits because of being a probiotic and due to the presence of molecules with biological activity. In this work, we have profiled the peptide composition of goat milk kefir at three different fermentation times using a peptidomics approach, in order to study changes in peptide concentrations and patterns of protein digestion throughout the fermentation time. We identified 2328 unique peptides corresponding to 22 protein annotations, with a maximum of peptides found after 24 h fermentation. We established different digestion patterns according to the nature of the proteins, and quantified the changes in the peptides appearing in all the fermentation times. We also identified 11 peptides that matched exactly to sequences with biological activity in databases, almost all of them belonging to caseins. This is the most comprehensive proteomic analysis of goat milk kefir to date.

Proteomics Analysis Reveals the Implications of Cytoskeleton and Mitochondria in the Response of the Rat Brain to Starvation.

Long-term starvation provokes a metabolic response in the brain to adapt to the lack of nutrient intake and to maintain the physiology of this organ. Here, we study the changes in the global proteomic profile of the rat brain after a seven-day period of food deprivation, to further our understanding of the biochemical and cellular mechanisms underlying the situations without food. We have used two-dimensional electrophoresis followed by mass spectrometry (2D-MS) in order to identify proteins differentially expressed during prolonged food deprivation. After the comparison of the protein profiles, 22 brain proteins were found with altered expression. Analysis by peptide mass fingerprinting and MS/MS (matrix-assisted laser desorption-ionization-time of flight mass spectrometer, MALDI-TOF/TOF) enabled the identification of 14 proteins differentially expressed that were divided into 3 categories: (1) energy catabolism and mitochondrial proteins; (2) chaperone proteins; and (3) cytoskeleton, exocytosis, and calcium. Changes in the expression of six proteins, identified by the 2D-MS proteomics procedure, were corroborated by a nanoliquid chromatography-mass spectrometry proteomics procedure (nLC-MS). Our results show that long-term starvation compromises essential functions of the brain related with energetic metabolism, synapsis, and the transmission of nervous impulse.

Valorisation of the microalgae Nannochloropsis gaditana biomass by proteomic approach in the context of circular economy.

Nannochloropsis gaditana is a non-flagellated microalgae that has been widely used for different purposes, mostly related with the industrial production of biofuels or aquiculture. However, in order to increase the economic viability of the obtained microalgae biomass from a production plant coupled to a coal power plant, a proteomic approach was initiated by using fresh and atomized microalgae samples, as the main used commercial forms. Above 51,000 high quality spectra were obtained per sample in the MS/MS analysis of whole proteome of N. gaditana, yielding above 7,500 peptides, leading the identification of 1,950 proteins, from the N. gaditana protein database, where 655 proteins were presented in all the replicates. The identified proteins were categorized according to gene ontology classification by molecular function and biological process. In this study, it has been described the first proteomic analysis of the microalgae N. gaditana under industrial conditions containing an important number of identified proteins. A significative presence of proteins with a potential role in different agri-food and biomedical applications was detected and studied being the core of future N. gaditana research to expand the current biotechnological applications of this microalga. SIGNIFICANCE OF THE STUDY: Three quarters of the planet earth correspond to seas and oceans, however its potential biotechnological use is still unknown. We described the first proteomic description of the microalgae N. gaditana under industrial conditions. Following the spirit of the EU initiatives of blue growth and the statements of circular economy, CO2 waste from a coal plant power has been transformed in a resource for microalgae biomass production, common product presentations were evaluated by proteomic, and its potential use of identified proteins in Agri-food and Biomedicine has been revealed.

Exploring the Denitrification Proteome of Paracoccus denitrificans PD1222.

Denitrification is a respiratory process that produces nitrous oxide as an intermediate, which may escape to the atmosphere before its reduction to dinitrogen through the nitrous oxide reductase NosZ. In this work, the denitrification process carried out by Paracoccus denitrificans PD1222 has been explored through a quantitative proteomic analysis. Under anaerobic conditions, with nitrate as sole nitrogen source, the synthesis of all the enzymes involved in denitrification, the respiratory nitrate, nitrite, nitric oxide, and nitrous oxide reductases, was increased. However, the periplasmic and assimilatory nitrate reductases decreased. Synthesis of transporters for alcohols, D-methionine, sulfate and copper, most of the enzymes involved in the tricarboxylic acid cycle, and proteins involved in other metabolic processes like lysine catabolism, fatty acids degradation and acetyl-CoA synthesis, was increased during denitrification in P. denitrificans PD1222. As consequence, an enhanced production of the central metabolite acetyl-CoA was observed. After establishing the key features of the denitrification proteome, its changes by the influence of a competitive electron acceptor, oxygen, or competitive nitrogen source, ammonium, were evaluated.

Alterations in oxidative responses and post-translational modification caused by p,p´-DDE in Mus spretus testes reveal Cys oxidation status in proteins related to cell-redox homeostasis and male fertility.

The major derivate of DDT, 1,1-dichloro-2,2-bis (p-chlorophenyl) ethylene (p,p´-DDE), is a persistent pollutant previously associated with oxidative stress. Additionally, p,p´-DDE has been linked to several metabolic alterations related to sexual function in rodents. In this study, we analysed the effects of a non-lethal p,p´-DDE dose to Mus spretus mice in testes, focusing on oxidative damage to biomolecules, defence mechanisms against oxidative stress and post-translational protein modifications. No increase in lipid or DNA oxidation was observed, although antioxidative enzymatic defences and redox status of glutathione were altered in several ways. Global protein carbonylation and phosphorylation were significantly reduced in testes from p,p´-DDE-exposed mice; however, the total redox state of Cys thiols did not exhibit a defined pattern. We analysed the reversible redox state of specific Cys residues in detail with differential isotopic labelling and a shotgun labelling-based MS/MS proteomic approach for identification and quantification of altered peptides. Our results show that Cys residues are significantly affected by p,p´-DDE in several proteins related to oxidative stress and/or male fertility, particularly those participating in fertilization, sperm capacitation and blood coagulation. These molecular changes could explain the sexual abnormalities previously described in p,p´-DDE exposed organisms.

Proteomic profile of the skin mucus of farmed gilthead seabream (Sparus aurata).

Fish skin mucus is the first line of defense against infections and it discriminates between pathogenic and commensal bacterial strains. Mucus composition varies amongst fish species and is influenced by endogenous and exogenous factors. This study describes the first proteome map of the epidermal mucus of farmed gilthead seabream (Sparus aurata). We used an integrative proteomic approach by combining a label-free procedure (LC-MS/MS) with the classical 2-DE-PMF-MS/MS methodology. The identified mucosal proteins were clustered in four groups according to their biological functions. Structural proteins (actins, keratins, tubulins, tropomyosin, cofilin-2 and filamin-A) and metabolic proteins (ribosomal proteins, proteasomal subunits, NACA, VCP, histones, NDPK, transferrin, glycolytic enzymes, ATP synthase components, beta-globin, Apo-A1 and FABP7) were the best represented functional categories. We also found proteins involved in stress response (WAP65, HSPC70, Cu,Zn-SOD, and PRDX1 and PRDX2) and signal transduction (PP2A 65kDa regulatory subunit, 14-3-3 protein beta/alpha, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, RhoGDI and PEBP1). Most of the identified proteins address different aspects of the innate immune response. Additionally, we analyzed bacterial peptides identified in the skin mucus of healthy S. aurata. These results revealed that genera belonging to the Lactobacillales order constitute the most abundant microorganism populations in this habitat. BIOLOGICAL SIGNIFICANCE: This work shows that proteomic methods can be used to characterize fish skin mucus. Using a coupled approach of LC-MS/MS and a 2-DE-PMF-MS/MS, we have obtained the first comprehensive view of the skin mucosal proteome of S. aurata, a fish species that is economically relevant for Mediterranean aquaculture. We identified a panel of proteins involved in a variety of biological functions, particularly in the innate immune response. Furthermore, to our knowledge, this is the first time a proteomic approach has been used to examine the microbiota in the skin mucus of a fish species. Overall, these results support further immunological researches in S. aurata and are relevant for the culture of this important fish species.

Identification of proteins containing redox-sensitive thiols after PRDX1, PRDX3 and GCLC silencing and/or glucose oxidase treatment in Hepa 1-6 cells.

The oxidation and reduction of cysteine thiols are thought to be a major mechanism for redox regulation. The aim of this study was to identify proteins with reactive thiols and determine their oxidation profiles under oxidative stress induced by simultaneous silencing of antioxidant defences (peroxiredoxin-1, peroxiredoxin-3, and the catalytic subunit of the glutamate-cysteine ligase), and/or treatment with glucose oxidase (GO). Using an approach that combined the labelling of reversibly oxidised cysteines, 2-DE protein separation and MS analysis, we identified 26 proteins with cysteines prone to reversible oxidation belonging to different functional classes. Among these proteins are those that have not been previously recognised as reversible oxidation targets, including cytoplasmic aspartate aminotransferase, proteasome subunit alpha type-6, heterogeneous nuclear ribonucleoproteins isoA2/B1 and A/B, and histidine triad nucleotide-binding protein 1. We provide the first evidence of reversible oxidation for specific cysteines, including Cys112 and Cys146 in glutamate dehydrogenase 1, Cys17 in actins, Cys5 in protein disulfide-isomerase A3, and Cys267 in the heat shock cognate 71 kDa protein. Silencing induced lower oxidative stress than GO treatment. Nevertheless, we detected some proteins particularly sensitive to oxidation by silencing. We hypothesised that these proteins may play a role in regulatory mechanisms by redox stress.

Proteomics in the liver of gilthead sea bream (Sparus aurata) to elucidate the cellular response induced by the intake of maslinic acid.

Maslinic acid (MA) is a pentacyclic triterpene used as a feed additive to stimulate growth, protein-turnover rates, and hyperplasia in fish. To further our understanding of cellular mechanisms underlying the action of MA, we have used 2-DE coupled with MS to identify proteins differentially expressed in the livers of juvenile gilthead sea bream (Sparus aurata) grown under fish-farm conditions and fed with a 100 mg/kg MA-enriched diet (MA(100)). After the comparison of the protein profiles from MA(100) fed fish and from control, 49 protein spots were found to be altered in abundance (≥2-fold). Analysis by MALDI-TOF/TOF allowed the unambiguous identification of 29 spots, corresponding to 19 different proteins. These proteins were: phosphoglucomutase, phosphoglucose isomerase, S-adenosyl methionine-dependent methyltransferase class I, aldehyde dehydrogenase, catalase, 6-phosphogluconate dehydrogenase, fumarylacetoacetate hydrolase, 4-hydroxyphenylpyruvic dioxygenase, methylmalonate-semialdehyde dehydrogenase, lysozyme, urate oxidase, elongation factor 2, 60 kDa heat-shock protein, 58 kDa glucose-regulated protein, cytokeratin E7, type-II keratin, intermediate filament proteins, 17-ß-hydroxysteroid dehydrogenase type 4, and kinase suppressor of Ras1. Western blot analysis of kinase suppressor of Ras1, glucose 6-phosphate dehydrogenase, elongation factor 2, 60 kDa heat-shock protein, and catalase supported the proteome evidence. Based on the changes found in the protein-expression levels of these proteins, we proposed a cellular-signalling pathway to explain the hepatic-cell response to the intake of a diet containing MA.

Proteomics in free-living Mus spretus to monitor terrestrial ecosystems.

We evaluated the suitability of high-throughput proteomic methods to monitor terrestrial ecosystems. Free-living Mus spretus from three sites along the "Domingo Rubio" (DR) stream were compared with mice from Doñana Biological Reserve ("Santa Olalla" lagoon (SOL) negative control), using specimens from an industrial settlement (phosphogypsum stacks (PS)) and rice fields ("Matochal" rice fields (ARZ)) as positive controls. Our 2-DE analysis showed 36 spots with significantly altered expression. Sixteen were identified by MALDI-TOF-PMF and peptide matching with Mus musculus databases. Identified proteins play different roles: cytoskeletal dynamics, proteolysis, biotransformation, oxidative-stress adaptation, and metabolism. Animals from different polluted environments showed contrasting differences in their proteomes, with specific increases and decreases in selected groups of proteins that seem to be co-ordinately regulated. Proteomic data were consistent with metal biomonitoring and conventional biomarker responses, indicating that DR (and PS/ARZ) animals sustained a heavier pollutant burden than SOL specimens and suffered a chronic oxidative stress. Whereas some protein expression differences may protect mice from pollutant toxicity, others should make them more susceptible. Transcript expression signatures agree with the documented lack of correlation between mRNA and protein levels. Nonetheless, a positive significant correlation was found between the gpx1 mRNA molecules and the intensity of one of the two identified GPX1 isospots.